Acute vascular diseases, such as myocardial infarction, stroke, pulmonary embolism, deep vein thrombosis, peripheral arterial occlusion and other blood system thromboses constitute major health risks. Such diseases are caused by either partial or total occlusion of a blood vessel by a blood clot, which consists of one or both of fibrin and aggregated platelets.
Blood platelets play an essential role in normal hemostasis. They have both a distinct hemostatic function and a thromboplastic function. Hemostasis is initiated within a few seconds following a trauma, when platelets begin to adhere to the edges of the lesion. This initial adherence of platelets may be mediated by collagen exposed at the site of blood vessel wall trauma or by newly generated thrombin. Once in contact with collagen or thrombin, platelets undergo activation (release reaction) releasing a variety of chemicals, including ADP and thromboxane A.sub.2. The released ADP and thromboxane A.sub.2 cause additional platelets from the issuing blood to aggregate to those already attached to the vessel wall. The newly attached platelets also undergo the release reaction and the process continues until a hemostatic platelet plug forms. In addition to releasing ADP and thromboxane A.sub.2, platelets also expose platelet factor 3, which promotes the clotting cascade, ultimately resulting in thrombin generation and fibrin deposition at the site of injury. Thrombin also binds to receptors on the platelet membrane and causes further platelet aggregation and release. The ultimate result is a mixed clot composed of aggregated platelets and polymerized fibrin.
Although necessary for normal hemostasis, clot formation resulting from platelet aggregation and release reaction is also responsible for a variety of life-threatening vascular diseases, such as myocardial infarction, stroke, peripheral arterial occlusion and other blood system thromboses. In patients suffering from such diseases, platelet aggregation is an undesirable event which should be inhibited. Inhibition of platelet aggregation may also be desirable in extracorporeal treatments of blood, such as dialysis, storage of platelets in platelet concentrates and following certain surgical procedures, such as heart-lung bypass.
During dialysis treatments, platelets tend to adhere to and aggregate on the walls of the dialysis membrane. This tends to reduce the efficiency of the treatment, as well as deplete platelets from the treated blood. In the case of platelet storage, platelet concentrates often undergo "storage lesion", a degradative process by which platelets are either activated or damaged. The practical result of such lesions is decreased storage life. Thrombin, which is generated in these platelet concentrates, is responsible for this degradation [A. P. Bode and D. T. Miller, "Generation and Degradation of Fibrinopeptide A in Stored Platelet Concentrates", Vox Sang., 51, pp. 192-96 (1986)]. In vascular surgery, the inhibition of blood clotting is essential for maintaining the integrity of the blood vessel treated. If clot formation occurs too soon after such surgery, it may threaten the life of the patient.
Although the mechanism of thrombin-induced platelet activation is poorly understood, it is believed to involve two steps: 1) binding of thrombin to a receptor on the platelet surface; and 2) thrombin-catalyzed proteolysis of a platelet surface substrate. A high-affinity thrombin binding site and a moderate-affinity thrombin binding site have been identified on the platelet surface and both are believed to be of physiological relevance [J. T. Harmon and G. A. Jamieson, "The Glycocalcin Portion of Platelet Glycoprotein Ib Expresses Both High and Moderate Affinity Receptor Sites for Thrombin", J. Biol. Chem., 261, pp. 3224-3229 (1986); J. T. Harmon and G. A. Jamieson, "Platelet Activation by Thrombin in the Absence of the High-Affinity Thrombin Receptor", Biochemistry, 27, pp. 2151-2157 (1988)].
The use of chemically modified and inhibitor-treated thrombins has demonstrated that both the binding step and the proteolysis step are components of platelet activation. For example, although thrombin treated with either hirudin or dansylarginine N-(3-ethyl-1-5-pentanediyl)amide (DAPA) cannot activate platelets, only the hirudin-treated thrombin fails to bind to the platelet thrombin receptor(s) [C. L. Knupp, "Effect of Thrombin Inhibitors on Thrombin-Induced Platelet Release and Aggregation", Thombosis Res., 49, pp. 23-36 (1988)]. Nevertheless, the inhibition of thrombin binding to platelet receptors has not been demonstrated to be sufficient to block thrombin-induced platelet activation. In fact, while hirudin blocks binding of thrombin to the platelet surface, it also inhibits the amidolytic function of the enzyme P. Walsmann and F. Markwardt, "Biochemical and Pharmacological Aspects of the Thrombin Inhibitor Hirudin", Pharmazie, 36, pp. 653-660 (1981)].
Current methods for treatment and prophylaxis of thrombotic diseases involve therapeutics which act in one of two different ways. The first type of therapeutic inhibits thrombin activity or thrombin formation, thus preventing clot formation. The second category of therapeutic accelerates thrombolysis and dissolves the blood clot, thereby removing it from the blood vessel and unblocking the flow of blood [J. P. Cazenave et al., Agents Action, 15, Suppl., pp. 24-49 (1984)].
Heparin, a compound of the former class, has been used widely to treat conditions, such as venous thromboembolism, in which thrombin activity is responsible for the development or expansion of a thrombus. Heparin exerts its effects by activating antithrombin III, a protein which complexes with and inactivates thrombin. Because of its mode of action, heparin is useful to prevent only thrombin-induced platelet aggregation. Even in that application, however, the overall efficacy of heparin is questionable. Furthermore, heparin produces many undesirable side effects, including hemorrhaging and heparin-induced thrombocytopenia. Moreover, in patients suffering from heparin-induced thrombocytopenia, an immune-mediated thrombocytopenia that may have dire thrombotic consequences, heparin actually accelerates platelet aggregation, often with fatal consequences. In other patients, such as those having an anti-thrombin III deficiency, heparin is simply less effective. Accordingly, the need exists for alternatives to conventional heparin-based therapies.
Hirudin is a naturally occurring polypeptide which is produced by the blood sucking leech Hirudo medicinalis. This compound, which is produced in the salivary gland of the leech, is the most potent natural inhibitor of coagulation known. Hirudin prevents blood from coagulating by binding tightly to thrombin (K.sub.d .about.2.times.10.sup.-11 M) in a 1:1 stoichiometric complex [S. R. Stone and J. Hofsteenge, "Kinetics of the Inhibition of Thrombin by Hirudin", Biochemistry, 25, pp. 4622-28 (1986)]. This, in turn, inhibits thrombin from catalyzing the conversion of fibrinogen to fibrin (clot).
The actual binding between hirudin and thrombin is a two-step process. Initially, hirudin binds to a "low" affinity site on the thrombin molecule (K.sub.d .about.1.times.10.sup.-8 M) which is separate from the catalytic site. Following the low affinity binding, hirudin undergoes a conformational change and then binds to the "high" affinity site on thrombin. This latter site corresponds to the active site of thrombin.
Hirudin has been shown to inhibit both the binding of thrombin to platelets [P. Ganguly and W. J. Sonnichsen, "Binding of Thrombin to Human Platelets and its Possible Significance", Br. J. Haemotol., 34, pp. 291-301 (1976); S. W. Tam and T. C. Detwiler, "Binding of Thrombin to Human Platelet Plasma Membrane", Biochim. Biophys. Acta, 543, pp. 194-201 (1978) and thrombin-induced platelet aggregation [S. W. Tam et al., "Dissociation of Thrombin From Platelets by Hirudin: Evidence for Receptor Processing", J. Biol. Chem., 254, pp. 8723-25 (1979)]. Therefore, hirudin has been viewed as a potential antiplatelet agent. However, several drawbacks are associated with hirudin use, including high cost, potential antigenicity and hemorrhaging.
The isolation, purification and chemical composition of hirudin are known in the art [P. Walsmann and F. Markwardt, "Biochemical and Pharmacological Aspects of the Thrombin Inhibitor Hirudin", Pharmazie, 36, pp. 653-60 (1981)]. More recently, the complete amino acid sequence of the polypeptide has been elucidated [J. Dodt et al. "The Complete Covalent Structure of Hirudin: Localization of the Disulfide Bonds", Biol. Chem. Hoppe-Seyler, 366, pp. 379-85 (1985); S. J. T. Mao et al., "Rapid Purification and Revised Amino Terminal Sequence of Hirudin: A Specific Thrombin Inhibitor of the Blood-Sucking Leech", Anal. Biochem, 161, pp. 514-18 (1987); and R. P. Harvey et al., "Cloning and Expression of a cDNA Coding for the Anti-Coagulant Hirudin from the Bloodsucking Leech, Hirudo medicinalis", Proc. Natl. Acad. Sci. USA, 83, pp. 1084-88 (1986)].
At least two different isospecific forms of hirudin, HV-1 and HV-2, have been sequenced and have been shown to differ slightly in amino acid sequence [R. P. Harvey et al., supra]. Both forms of hirudin comprise a single polypeptide chain protein containing 65 amino acids in which the amino terminus primarily comprises hydrophobic amino acids and the carboxy terminus typically comprises polar amino acids. More specifically, all forms of hirudin are characterized by an N-terminal domain (residues 1-39) stabilized by three disulfide bridges in a 1-2, 3-5, and 4-6 half-cysteinyl pattern and a highly acidic C-terminal segment (residues 40-65). In addition, the C-terminal segment of hirudin is characterized by the presence of a tyrosine residue at amino acid position 63 which is sulfated.
In animal studies, hirudin, purified from leeches, has demonstrated efficacy in preventing venous thrombosis, vascular shunt occlusion and thrombin-induced disseminated intravascular coagulation. In addition, hirudin exhibits low toxicity, little or no antigenicity and a very short clearance time from circulation [F. Markwardt et al., "Pharmacological Studies on the Antithrombotic Action of irudin in Experimental Animals", Thromb. Haemostasis, 47, pp. 226-29 (1982)].
Despite hirudin's effectiveness, however, studies have shown that hirudin prolongs bleeding time in a dose-dependent manner, thus making the determination and administration of proper dosages critically important. Furthermore, the high cost and low supply of the naturally occurring product has prevented its widespread use.
In an effort to create a greater supply of hirudin, attempts have been made to produce the polypeptide through recombinant DNA techniques. The presence of an O-sulfated tyrosine residue on native hirudin and the inability of microorganisms to perform a similar protein modification made the prospect of recombinant production of biologically active hirudin highly speculative. The observation that desulfato-hirudins were almost as active as their sulfated counterparts [U.S. Pat. No. 4,654,302], however, led the way to the cloning and expression of hirudin in E.coli [European patent applications 158,564, 168,342 and 171,024] and yeast [European patent application 200,655]. Despite these advances, hirudin is still somewhat expensive to produce and it is not widely available commercially.
Recently, efforts have been made to identify peptide fragments of native hirudin which are also effective in lowering clotting time. An unsulfated 21 amino acid C-terminal fragment of hirudin, N.sup..alpha. -acetylhirudin.sub.45-65, inhibits clot formation in vitro. In addition, several other smaller, unsulfated peptides corresponding to the C-terminal 11 or 12 amino acids of hirudin (residues 55-65 and 54-65) have also demonstrated efficacy in inhibiting clot formation in vitro [J. L. Krstenansky et al., "Antithrombin Properties of C-terminus of Hirudin Using Synthetic Unsulfated N.sup..alpha. -acetyl-hirudin.sub.45-65 ", FEBS Lett, 211, pp. 10-16 (1987)]. Such peptide fragments, however, may not be fully satisfactory to dissolve blood clots in on-going therapy regimens. For example, N.sup..alpha. -acetylhirudin.sub.45-65 has a specific activity four orders of magnitude lower than native hirudin.
Accordingly, the need still exists for an effective inhibitor of both clot formation and platelet aggregation and secretion that is not characterized by the side effects associated with conventional agents and which can be produced in commercially feasible amounts.